Introduction: Glycated Hb (HbA1c) has humongous role both in diagnosis as well as in guiding the clinicians in making therapeutic decision in diabetic patients. There are various methods of laboratory estimation of glycated Hb depending on charge and affinity. However, the methods are affected by a number of factors like Hb variants, drugs, erythrocyte survival. Aim: To identify and evaluate abnormal peaks in chromatogram of High Performance Liquid Chromatog. (HPLC) and compare it with boronate affinity chromatog. and it also assessed the interference of elution of silent Hb variants or drugs in exactness of HbA1c estimation by HPLC. Materials and Methods: A cross-sectional survey was conducted in the laboratory of Purwanchal Nagarik Samity, Kolkata, West Bengal, India, from Nov. 2020 till August 2021, where, 2500 samples were evaluated for HbA1c estimation Abnormal variant window, unknown peaks or spuriously low HbA1c were identified in eight samples, where estimation was done by HPLC. These were processed further to investigate for the Hb variants. The glycated Hb in those samples was further estimated by Boronate affinity chromatog. Results: Out of the eight samples, three patients were Hb E trait, two were Hb E homozygous and one had Sickel cell trait. However, two samples showed a normal chromatogram in HbA2/HbF mode. The glycated Hb was affected by Hb E, sickel cell traits as well as other interferences that may cause inappropriate lowering of glycated Hb. Conclusion: Extensive literature review recommends that progression of various micro/macro-vascular complications in diabetic individuals can be arrested by maintaining normal HbA1c levels. Therefore, the laboratory consultant should meticulously take into consideration factors like plasma glucose concentration, drug history, biol. variation or abnormal haemoglobinopathies. Thus, every laboratory catering to the huge burden of diabetic patients, where the prevalence of haemoglobinopathy is high, should individualise the method of HbA1c estimation